Bioluminescence Chemical Principles

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u m i n e s c e n c e

C h e m i c a l P r i n c i p l e s a n d M e t h o d s

O s a m u S h i m o m u r a

F o r m e r ly S e n i o r S c i e n t i s t a t th e M a r in e B i o l o g i c a l L a b o r a t o r y

W o o d s H o l e M a s s a c h u s e t t s

fe World Scientific

EY L O N D O N S I N G A P O R E B E I J I N G S H A N G H A I H O N G K O N G T A I P E I C H E N N A I


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Published by

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Library of Congress Cataloging-in-Publication Data

Shimomura, Osamu 1 928 -

Bioluminescence : chemical principles and methods / Osamu Shimomura

p. cm.

Includes bibliographical references (p. ).

ISBN 981-256-801-8

1.

Bioluminescence. 2. Chem iluminescence. I. Title.

QH641.S52 2006

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Preface

In studying the chemical aspects of bioluminescence, comprehen

sive reviews of practical use were scarce in the past except on lumi

nous bacteria. This is a considerable inconvenience and disadvantage

to researchers. In fact, I have been frequently frustrated myself by

the need to search for old articles published 30^10 years ago to find

data on some basic properties of bioluminescent substances, such as

the absorption spectra and luminescence activities of luciferins. In the

absence of any compendium of the substances and reactions involved

in bioluminescence, researchers will have to spend their precious time

delving thro ug h the li terature in ord er to find the needed inform ation .

Such a situation may discourage new investigators who are interested

in the chemical study of bioluminescence, and might hamper them

from actually taking up a project. Upon consideration of these mat

ters,

I decided to write this book.

The present book describes all the significant studies and findings

on the chem istry of the mo re tha n 3 0 different biolum inescent systems

presently known, accompanied by over 1000 selected references. It

includes descriptions of the purification and properties of biolumines

cent compounds, such as luciferins, luciferases and photoproteins, and

the mechanisms of luminescence react ions. To make the book more

useful th an a mere review volum e an d to save researchers t ime in look

ing into original references, I have included a considerable amount

of original experimental methods, data and graphs. In addit ion, I

have included some new data and experimental methods unavail

able elsewhere. I hope this volume will be useful to researchers and

students, and it will be my greatest pleasure if this book contributes


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vi Bioluminesce nce: Che mica l Principles and Metho ds

to the finding of new luciferin structures and new luminescence

mechanisms.

I am grateful to J. Woodland Hastings, Satoshi Inouye and

Yoshihiro Ohmiya who kindly read a draft version of this book

and provided me with valuable suggestions and advice. I also would

like to express my sincere thanks to Steven Haddock, John Brinegar

and Sachi Shimomura for their help in correcting my English, and

Sook Cheng Lim for editing this book.

I have been extremely fortunate to be able to continue my

research on bioluminescence for 50 years without interruption. It was

made possible with the help of many people and the continued sup

port from the National Science Foundation; I am particularly indebted

to Toshio Goto, Yoshito Kishi , Benjamin Kaminer and J . Woodland

Hastings for their kind help. All my work has stemmed, however,

from the initiatives taken by my three mentors: the late Professors

Shungo Yasunaga (Nagasaki Universi ty) , Yoshimasa Hirata (Nagoya

University) and Frank H. Johnson (Princeton University). Yasunaga,

in 1955, advised me to shift my specialty from pharmacy to chem

istry and he arranged for me to work at Hirata 's organic chem

istry lab; Hirata gave me the very difficult problem of crystallizing

Cypridina luciferin, which eventually rewarded me with the experi

ence and k now ledge necessary as a researcher; and Joh nso n, in 1 96 1,

gave me the subject ofAequorea an d helped me for 20 years in solving

the problems of aequorin and other bioluminescent substances.

With my great respect, I dedicate this book to the memory of my

three mentors .

Osamu Shimomura


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Contents

Preface

v

A bbreviations, Symbols and D efinitions xv

Introduction xvii

The Beginning of the Chemical Study

of Biolum inescence xix

Luciferin xx

Photoprotein xxi

Chemical Studies on Bioluminescence in the Last One

H und red Years xxi i

Chem ical Study of Bioluminescence in the Fu ture xxiv

Th e C onte nts of this Book xxvi

1 Th e F ireflies and Lu m inous Insects

1

1.1 T he Fireflies 3

1.1.1 Essen tial Fa cto rs in the Firefly Lum inescence

Reaction 3

1.1.2 Firefly Luciferin an d O xylu ciferin 5

1.1.3 Firefly Lu ciferase 8

1.1.4 Assays of Luciferase Activity, AT P and

Luciferin 11

1.1.5 Ge neral Ch aracteristics of the

Bio lum inescen ce of Fireflies 12

1.1 .6 M echanisms of the Bio luminescence . . . . 15

vii


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viii Bioluminescence: Chem ical Principles and Me thods

1.1.7 Ligh t Em itters in the Firefly Lum inescence

System 17

1.1.8 A N ote on the D ioxe tano ne Pathw ay and the

l s

O-in corp ora t ion Exper iment 19

1.2 Phe ngo didae and Elateroidae 23

1.2.1 Phe ngo didae 24

1.2.2 Ela teridae 24

1.3 D iptera 2 5

1.3.1 The Glow -wo rm

Arachnocampa

25

1.3.2 The Am erican Glow -worm

Orfelia

2 7

2 Lum inous Bacteria

30

2.1 Fac tors Re quired for Bioluminescence 31

2.2 Bacterial Luciferase 33

2.3 Lon g-chain Aldehyde 35

2.4 M echan ism of Luminescence Reac tion 37

2.5 Assay of Luciferase Activity 39

2.6 Q ua ntu m Yield of Long-chain Aldehydes 4 1

2.7 In vivo Luminescence of Luminous Bacteria . . . . 41

3 Th e OstracodC ypridina (Vargula) and Other

Lum inous Crustaceans

47

3.1 The Ostraco d

Cypridina

49

3.1.1 Overview of Ostrac od a 49

3.1.2

Cypridina kilgendorfii

M iiller 51

3.1.3 Research on

Cypridina

Luminescence

before 195 5 53

3.1.4 Purification an d Cry stallization of

Cypridina

Luciferin 55

3.1 .5 Prop erties of

Cypridina

Luciferin 58

3.1.6 Ox yluciferin an d Etioluciferin 62

3.1.7 Purification and M olecu lar Properties

of

Cypridina

Luciferase 62

3.1.8 Luciferin-luciferase Lum inescence

Reaction 64

3.1.9 Q ua ntu m Yield 69


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Contents ix

3.2 Euphausi ids Euphausia pacifica and

Meganyctiphanes norvegica 71

3.2.1 Involvement of the Fluorescent C om po un d F

an d Protein P 71

3.2.2 Fluorescent C om po un d F 74

3.2.3 Protein P 79

3.2.4 Lum inescence Re action 80

3.3 The De capod Shrimp Op lophorus gracilirostris . . 82

3.3.1 Oplophorus Luciferase 82

3.3.2 Coelenterazine-luciferase Re action 83

3.4 Cop epoda 88

Th e JellyfishAequorea and Other L um inous C oelenterates 9 0

4.1 The Hy drozoan M edusa Aequorea aequorea . . . . 92

4.1 .1 H istory of the Biochemical Study of

Aequorea Bioluminescence 94

4.1 .2 Ex tractio n an d Purification of A equ orin . . 95

4.1 .3 Properties of A equ orin 100

4.1 .4 Discovery of the Co elenterazine M oiety

in Aequor in I l l

4.1.5 Reg enerat ion of Ae quorin from

Apoaequor in 113

4.1.6 Reco mb inant Aequo r in 116

4.1 .7 Semisynthetic Ae quorins 118

4.1.8 The In Vivo Luminescence of Aequorea . . 129

4.2 The Hy droid

Obelia

(Hydrozoan) 133

4.2.1 N atu ral Obelins 133

4.2.2 Reco m binant Obel in 134

4.3 The Hy drozoan M edusa Phialidium gregarium . . 137

4.4 O ther Bioluminescent H yd rozo an s 138

4.5 Th e Scyphozoans Pelagiaand Periphylla 140

4.5.1 Pelag ia noctiluca 140

4.5.2 Periphylla periphylla 140

4.6 The An thozoan

Renilla

(Sea Pansy ) 14 7

4.7 Green Fluorescent Protein (GFP) 15 1

4.8 The Ctenoph ores 154


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x B i o l u m i n e s c e n c e : C h e m i c a l P r in c ip le s a n d M e t h o d s

5 Th e Coelenterazines

159

5.1 Discovery of Co elenterazine 159

5.2 Oc currence of Co elenterazine 160

5.3 Prop erties of C oelen terazine an d its De rivatives . . 16 5

5.4 Ch em i- and Bioluminescence Re actions

of Co elenterazine 168

5.5 Chem ical Re actions of Co elenterazine 173

5.6 Synthesis of Co elen terazine s 17 6

5.7 Co elenterazine Luciferases 176

6 Lum inous Mollusca

180

6.1 The Limpet Latia 182

6.2 Th e Clam Pholas dactylus 192

6.3 Lum inous Squids (Ceph alopoda) 199

6.3 .1 The Firefly Squid Watasenia scintillans . . . 200

6.3.2 Th e Purpleb ack Flying Squid

Symplectoteuthis oualaniensis (Tob i-ika) . . 20 4

6.3.3 Th e Lu m inou s Flying Squid

Symplectoteuthis luminosa (Suji-ika) . . . . 2 1 0

7 Annelida

2 1 6

7.1 The Tu bew orm Chaetopterus variopedatus 216

7.1.1 Biochem istry of the Lum inescence

of

Chaetopterus variopedatus

218

7.1.2 Pro perties of the Chaetopterus Photoprotein

an d its Lum inescence Re action 2 21

7.2 Th e Bermuda Fireworm Odontosyllis 225

7.3 Lum inous Ea rthw orm s (Oligochaeta) 234

7.4 Polynoid Scaleworm Harm othoe lunulata 242

7.5 Th e Polychaete

Tomopteris

246

8 D inoflagella tes and O ther Protozoa

248

8.1 Rad iolarians 24 8


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Con ten ts x i

8.2 Dinoflagellates 24 9

8.2.1 Cu ltivation and H arve sting

of Dinoflagellates 2 5 1

8.2.2 Scintillons 2 5 1

8.2.3 Th e Luciferase of Gonyaulax polyedra . . . 25 2

8.2.4 Ex trac tion an d Purification of Dinoflagellate

Luciferin 256

8.2.5 Prop erties of Dinoflagellate Luciferin . . . . 2 58

8.2.6 Ch em ical Structu res of Dinoflagellate

Luciferin and its Oxidat ion Product s . . . . 260

8.2.7 Chem ical M ech anism of Dinoflagellate

Bioluminescence 263

8.2.8 Luciferin Binding Protein of

Dinoflagellates 264

9 Lum inous Fungi

2 6 6

9.1 An Ov erview on Fung al Bioluminescence 26 6

9.2 Early Studies on the Bioche mistry of

Lum inous Fungi 268

9.3 Role of Superoxide in Fungal Luminescence . . . . 271

9.4 Studies on

Pattellas stipticus

275

9.4.1 Panal 27 7

9.4.2 Activation Prod ucts of Pana l 27 9

9.4.3 PS-A andP S-B 282

9.4.4 A ctivation of PS-A an d PS-B 28 3

9.4.5 M echan ism of thein vivo Bioluminescence of

P. stipticus

289

9.4.6 Synthetic Studies of Pane llus Luciferin . . . 2 9 1

9.5 Studies on Mycena citricolor 294

9.5.1 Lucifer in Ob tained by K uw aba ra and

Wassink 294

9.5.2 Studies on theMycena citricolor

Luminescence by the Au thor 29 4

9.6 Sum ma ry on the Che m istry of Fung al

Luminescence 298


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xii Bioluminescence: Che mical Principles and Methods

10 O ther Lum inous Organisms

301

10.1 O ph iuro ide a: Britt le Stars 30 1

10.1.1 The Brittle Star Ophiopsila californica . . . 302

10.1.2 The Brittle Star

Amphiur'a fillformis

. . . . 3 0 7

10.2 Millipede Luminodesmus sequoiae (Diplopo da) . . 30 7

10.3 Centipede Orphaneus brevilabiatus (Chilopod a) . . 31 4

10.4 Hem icordata 315

10.4.1 The Acorn Worm Balanoglossus

biminiensis 315

10.4.2 The Luciferin of Ptychodera flava 318

10.5 Tun icates (Phylum Ch orda ta) 319

10.6 The Lum inous Fishes 322

10.6.1 Coastal and Shallow-water Fishes that

Utilize Cypridina Luciferin 32 3

10.6.2 Oce anic Deep-sea Lum inous Fishes 32 7

10.6.3 Future Research on Fish

Bioluminescence 330

Appendix

333

A T ax on om ic Classification of Selected Lu m inou s

Organisms 333

B Lists of Luciferins, Luciferases an d Ph oto pro tein s

Isolated 340

C M iscellaneou s Techn ical Inform ation 34 9

C I Basic Principle of the Isolation of

Bioluminescent Substances 34 9

C 1.1 Reversible Inhibition of

Bioluminescence 350

C I. 2 E xtra ctio n of Luciferin-luciferase

Systems 353

C I .3 Solubilization of Proteins 35 3

C1 .4 Purification 35 5

C2 Storage of Sam ples 35 6

C3 M easurem ent of Luminescence 360


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Con ten ts x i i i

C4 Ca librat ion of Lum inom eter and the

M easurem ent of Q ua ntu m Yield 361

C5 M easurem ents of Coe lenterazine, i ts

Derivat ives, and o ther Im po rtant Substances

in Bioluminescence 36 3

C 5.1 Assay of Co elenterazine 36 3

C5 .2 Assay of the Co elenterazine

Luciferase Ac tivity 36 4

C 5.3 Assay of the Stabilized Fo rm s

of Co elenterazine 36 5

C5 .4 Assay of De hydrocoe lenterazine . . 36 6

C 5.5 Assay of Cypridina L uciferin . . . . 3 66

C5 .6 Assay of Cypridina L uciferase . . . . 3 6 7

C 5.7 Assay of Ca

2+

-sensitive

Photoproteins 368

C5 .8 M easuring Bioluminescence

in the Field 36 9

C 6

l s

O-Label ing of the React ion

P r oduc t C 0

2

370

C 7 Glass Blowing 37 4

D Advice to Students W h o are Interested in Studying

the Ch em istry of Bioluminescence 37 5

References 379

Index

455


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Abbreviations, Symbols and Definitions

8

A.

|xm Hg

A

ADA

AMP

ATP

Bis-tris

BSA

CAPS

CHES

CIEEL

CTAB

D a

DEAE

Diglyme

D M F

D M S O

D N A

D T T

E. coli

EDTA

EGTA

FAB

Molar absorption coefficient (absorbance of a 1M

solution in a

1

cm -pa th cell)

Wavelength

1/760,000 of one atmospheric pressure

A bso rban ce [log(Io/J)]; optical density (OD );

fo r 1 cm light path if not specified

N-(2-Acetamido)-2-iminodiacet ic acid

Adenosine 5 -monophosphate

Adenosine 5 -triphosphate

bis (2-Hy dr oxyethyl) am inotris (hydroxy m ethyl )metha ne

Bovine serum albumin

3 -(Cyclohe xylam ino)-1 -propan esulfonic acid

2-(Cyclohexylamino)ethanesulfonic acid

Chemically initiated electron-exchange luminescence

Hexadecyl t r imethylammonium bromide

Dalton (1/12 of the mass of one atom of

1 2

C )

Diethylaminoethyl

2-Methoxyethyl ether

Dimethylformamide

Dimethylsulfoxide

Deoxyribonucleic acid

Dithiothrei tol

Escherichia coli

Ethylenediaminetetraacetic acid

Ethyleneglycol-bis(2~aminoethylether)-

N,N,N' ,N'- te t raacet ic acid

Fas t a tom bombardment ionizat ion

X V


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xvi Bioluminesce nce: Chem ical Principles and Metho ds

FAD

F M N

F M N H

2

FPLC

F W H M

GFP

HEPES

HP LC

I

K

d

K

m

LBP

LCC

LU

m/z

MES

M O P S

M

r

NAD+

N A D H

N M R

N T A

PAGE

pCa

Pi

P^a

quan t um

yield

SB3-12

SDS

SOD

TAPS

TLC

Tris

UV

YC

Flavin adenine dinucleotide

Riboflavin 5 -monophosphate

Riboflavin 5'-monophosphate, reduced form

Fast protein l iquid chromatography (Pharmacia)

Full width at half maximum

Green fluorescent protein

N-(2-Hydroxyl)piperazine-N'-(2-ethanesulfonic acid)

High performance l iquid chromatography

Intensity

Dissociat ion constant

Michaelis constant

Luciferin binding protein

Lauroylcholine chloride

Light unit

Mass to charge ratio

2-(N-Morpholino)ethanesulfonic acid

3-(N-Morpholino)propanesulfonic acid

Relative molecular weight (dimensionless number)

Nicotine amide adenine dinucleotide, oxidized form

Nicotine amide adenine dinucleotide, reduced form

Nuclear magnetic resonance

Nitrilotriacetic acid

Polyacrylamide gel electrophoresis

Minus log of molar Ca

2 +

concentrat ion, log[Ca

2+

]

Isoelectric point

Minus log of acidic dissociation constant

The number of photons emit ted divided by the

number of molecules reacted

3-(Dodecyldimethylammonio)propanesulfonate

Sodium dodecylsulfate

Superoxide dismutase

N-[Tris(hydroxymethyl)methyl]-

3-aminopropanesulfonic acid

Thin- layer chromatography

Tr is (hy dr oxym ethyl) a m ino m etha ne

Ultraviolet

Yel low compound


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Introduction

The emission of l ight from animals and plants has inspired the

curiosity and interest of mankind ever since the ancient t imes of

A ristotle (3 8 4 -3 2 2 B.C.) an d Pliny (A.D. 2 3 -7 9 ). It ha s been the tar

get of investigations by a number of great naturalists, physicists and

physiologists (Harvey, 1957). Using a vacuum pump he buil t , Robert

Boyle (1627-1691) showed that the luminescence of meat and fungi

requires air . Benjamin Franklin (1706-1790), who found that l ight

ning is caused by electricity, hyp othesized th at the pho spho rescen ce of

the sea is an electrical phenomenon, but he did not hesitate to change

this op inion w hen he discovered th at the light in seaw ater cou ld be fil

tered off with a cloth. Pao lo Panceri (1 83 3- 18 77 ) is no ted for his p ub

lications on the anatomy and histology of various types of luminous

organism s, and Rap hael Dub ois (184 9-1 92 9) discovered lucifer in and

luciferase. The secrets of the chemistry of bioluminescence, however,

began to be uncovered only in the 20th century. Out of necessity,

Eilhardt W iedem ann (1888) had created the term lum inescence,

meaning the emission of cold light, and Harvey (1916) used the term

biolu m inesc enc e, luminescence from living orga nism s, possibly for

the first time.

Today, bioluminescence reactions are used as indispensable ana

lytical tools in various fields of science and technology. For example,

the firefly bioluminescence system is universally used as a method of

measuring ATP (adenosine triphosphate), a vital substance in living

cells; Ca

2 +

-sensitive photoproteins, such as aequorin from a jellyfish,

are widely util ized in monitoring the intracellular Ca

2 +

that regu

lates various important biological processes; and certain analogues

xvii


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19/498

Introduction xix

and fungi). There are also many that are intermediates of these two

groups. The bioluminescence system of an organism may involve a

series of interrelated chemical reactions, although the light is emit

ted only from the reaction that produces the singlet excited state of

light-emitter, a reaction termed light-emitting rea ctio n. The re are

various different light-emitting reactions, but all involve the oxidation

of a substrate (usually luciferin) that provides the energy for generat

ing an excited state. So far as is known, every bioluminescence reac

tion is basically a chemiluminescence reaction. It is remarkable that

animals and plants have developed their functional abilities of biolu

minescence, integrating the mechanisms of various disciplines such as

chemistry, physics , physiology and m orpho logy.

The Beginningofthe Ch em ical StudyofB iolum inescence

It is generally considered t ha t the m od ern study of bioluminescence

began when Dubois demonstrated the first example of a luciferin-

luciferase reaction in 1885. He made two aqueous extracts from the

lum inou s We st Indies beetlePyrophorus. O ne of the extrac ts w as pre

pared by crushing the light organs in cold water, resulting in a lumi

nous suspension. The luminescence gradually decreased and finally

disappeared. The other extract was prepared by initially treating the

light organs with hot water, which immediately quenched the light,

and then i t was cooled. The two extracts gave a luminescence when

mixed together . He found nearly the same phenomenon with the

extracts of the clam Pbolas dactylus (Dubois , 1887). Dubois con

cluded that the cold water extract contained a specific, heat labile

enzyme necessary for the light-emitting reaction, and introduced a

term luciferase for this enzym e. H e also con cluded th at the ho t

water extract, in which this enzyme has been destroyed by heat, con

tained a specific, relatively heat stable substance, which he designated

lucife rine (presently spelled luciferin). T h u s, the luciferin-luciferase

reaction can be viewed as a substrate-enzyme reaction that results in

the emission of light.

Following the discovery of luciferin and luciferase by Dubois,

the person who made the greatest contr ibution to the knowledge of


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x x B i o l u m i n e s c e n c e : C h e m i c a l P r in c ip l es a n d M e t h o d s

bioluminescence was E. Newton Harvey (1887-1959) of Princeton

University (biographical memoir: Johnson, 1967). He was initially a

physiologist, but was quickly captivated by the phenomenon of biolu

minescence, and his interest in bioluminescence grew into his lifelong

project. Harvey traveled widely and studied the bioluminescence of

a great variety of luminous organisms, producing over 300 publica

t ions.

He understood the underlying foundation of the chemistry of

bioluminescence reac tions, despite the fact that l it tle w as kno w n ab ou t

the actual chemical reactions at the time. His book Bioluminescence

published in 1952 is considered the bible of bioluminescence.

It was a common belief that all phenomena of bioluminescence

were caused by the luciferin-luciferase reaction until the biolumines-

cent protein aequorin was discovered in 1962 (Shimomura et al.,

19 62). W hen the term s luciferin a nd luciferase w ere found to be un suit

able for categorizing the two bioluminescent proteins, aequorin and

another from the tubeworm Cbaetopterus, a new term pho topro-

tein w as introd uce d to supp lem ent the term luciferin (Sh imom ura

and Johnson, 1966). Further explanations for the terms luciferin and

photoprotein are given below.

Luciferin

The term luciferin has never been strictly defined. Its precise

meaning has changed from time to time, and may change in the

future. Luciferin originally meant a relatively heat-stable, diffusible

substanc e existing in the cooled, ho t-w ate r extrac t of lum inou s org ans ,

as an essential ingredient needed for the emission of bioluminescence.

Harvey discovered that the luciferin of the clam Pholas differs from

that of the ostracod Cypridina (Harvey, 1920), and stated in his 1952

bo ok It is pr ob ab le tha t the luciferin or luciferase from a species in

one gro up may be quite different chemically from tha t in an ot he r. It

became ap pa ren t by the end of the 195 0s tha t the luciferins existing in

Cypridina, the fireflies and luminous bacteria are chemically different

from each other, generating a widely held view that the luciferins of

all luminous species are different except in species biologically closely

related. Ho w ev er, this view did not last long . A rou nd 19 60 , a luciferin


21/498

I n t r oduc t ion xx i

identical to the luciferin of Cypridina was discovered in the luminous

fishes Parapriacanthus and Apogon. M ore ov er, i t w as followed by a

series of discoveries during the period 1970-1980 that coelenterazine,

a luciferin, exists in a wide range of luminous organisms that include

various coelenterates, shrimps, squids and fishes.

Based on the presently available information, i t seems appropri

ate to define luciferin as the general term of an orga nic com po un d

that exists in a luminous organism and provides the energy for l ight

emission by being oxidized, normally in the presence of a specific

luciferase. The luciferase catalyzes the oxidative light-emitting reac

tion of the luciferin. It is an important criterion that a luciferin is

capable of emit t ing photons in proport ion to i ts amount under s tan

dardized condit ions.

A bioluminescence reaction of a luciferin is basically a chemilu-

minescence reaction. The luciferin is an absolute requirement as the

source of light energy, but the luciferase, an enzyme (protein), might

not be needed if its role could be replaced by other substance(s).

Recent studies at the author 's laboratory suggest that a luciferase

m ight no t be involved in the light-em itting reaction of lum inou s fungi

(see Chapter 9).

Photoprotein

In 1 9 6 1 , an unu sual bioluminescent protein w as discovered from

the jellyfish Aequorea, and it wa s nam ed aequorin (Shimom ura et al.,

1962). Aequorin emits l ight in aqueous solutions merely by the addi

t ion of C a

2 +

, regardless of the presence or absence of oxygen. The light

is emitted by an intramolecular reaction of the protein, and the total

light emission is proportional to the amount of the protein used. The

properties of aequorin do not conform to the definition of luciferin

or luciferase. Tentatively we thought aequorin could be an extraordi

nary excep tion occ urring in na ture . W e discovered, howev er, an othe r

example of bioluminescent protein in 1966 in the parchment tube-

w o r m Chaetopterus tha t emits l ight w hen a perox ide and Fe

2 +

are

added; the total l ight emission was proport ional to the amount of

the protein used, l ike aequorin. Considering the possibili ty of find

ing many similar bioluminescent proteins from luminous organisms,


22/498

xx i i B io lum inesc enc e : Ch em ica l P r inc ip les an d M e th od s

we prop osed a new term ph oto pro tein to designate these unu sual

bioluminescent proteins (Shimomura and Johnson, 1966).

Th us, ph oto pro tein is a general term of the biolum inescent pro

teins that occur in the light organ of luminous organisms and are

capable of emitting light in proportion to the amount of the protein

(Shimom ura, 19 85). A ph otop rotein could be an enzym e-substrate

complex that is more stable than its dissociated components, enzyme

and substrate. Due to its greater stabili ty, a photoprotein occurs as

the primary luminescent component in the light organs instead of i ts

dissociated components. In the light organs of the jellyfish Aequorea,

aequorin is highly stable as long as Ca

2 +

is absent, but its less stable

com pon ents , coelenterazine and a po aeq uo rin, are hardly detectable in

the jellyfish.

Several different types of photoprotein are presently known,

for example: the Ca

2+

-sensitive types found in various coelenter-

ates (aequorin, obelin, mnemiopsin) and protozoa (thalassicolin);

the peroxide-activation types found in scaleworm (polynoidin) and

the clam Pbolas (pholasin); and the ATP-activation type found in a

Sequoia millipede Luminodesmus.

Chemical Studies on Bioluminescence inthe Last O ne H undred Years

Bioluminescence is a com plicated p hen om eno n. A complete un der

standing of the phenomenon will require studies in a wide range

of disciplines, including morphology, cell biology, physiology, spec

troscopy, biochemistry, organic chemistry, and genetics. In the past

century, there have been very significant gains in the understanding of

bioluminescence in all these disciplines.

Important findings, discoveries and breakthroughs in chemistry

after the discovery of luciferin and luciferase by Dubois are chrono

logically listed in the table shown below. The chemical structures

of luciferins and light-producing groups have been determined and

the light-emitting reactions elucidated in considerable detail in the

bioluminescence of eight different types of organisms, namely, the

fireflies, the ostracod Cypridina, luminous bacteria, coelenterates, the

l impet Latia, earthworms, krill and dinoflagellates. A new concept of


23/498

I n t r oduc t ion xx i i i

Major Progress in Research into the Chemistry of Bioluminescence

Year Description

188 5 Discovery of luciferin-luciferase reaction

193 5 Benzoy lation of Cypridina luciferin

194 7 ATP requ irem ent in firefly luminescence

195 3 Re quire m ent for long-cha in aldehyd e (luciferin) in bacterial

luminescence

1954 FM N H 2 requirem ent in bacterial luminescence

195 7 Crystallization of Cypridina luciferin

19 57 Cry stalliz ation of firefly luciferin

1958 Cypridina luciferin in fishes; the first cross reaction discovered

19 61 -1 96 3 Structure of firefly luciferin

196 2 Discovery of aeq uor in and GFP (green fluorescent protein )

196 6 Structure of Cypridina luciferin

1966 Concept of photop rote in

196 8 Structure of Latia luciferin

19 67 -19 68 Dio xetano ne mechan ism propo sed in firefly and Cypridina

luminescence

1968 Reg ulation of dinoflagellate luminescence by p H

1971 Dio xetano ne mechan ism confirmed in Cypridina luminescence

197 4 Long -chain aldehydes identified in lum inous bacteria

19 75 Disco very of coe lente razin e (a luciferin)

1975 Structure of l ight-emitting chro m oph ore of aequo rin

1975 Regeneration of aequo rin

1976 Structure of earth w orm luciferin

1977

Renilla

luciferin identified to be coelenterazine

197 7 Dio xetan one mechanism confirmed in firefly luminescence

1978 Coelenterazine-2-hy droperoxide in aequ orin

1979 Structure of the chro m oph ore of GFP

1981 Structure of the luminescence autoindu cer of lum inous bacteria

19 84 -1 98 5 Firefly luciferase cloned

19 85 -1 98 6 Bacterial luciferase cloned

198 5-1 986 Apoaequ orin c loned

198 8 Stru cture of krill luciferin

1988 Semisynthetic aequo rins prepare d

19 89 Stru cture of dinoflagellate luciferin

1992 GFP cloned

19 94 GFP expres sed in living cells

199 6 Crysta l structu re of bacterial luciferase

19 96 Cr ysta l stru ctu re of firefly luciferase

1996 Crystal structure of GFP

20 00 Crystal structures of aequo rin and obelin

20 05 Crystal struc ture of dinoflagellate luciferase


24/498

xxiv Bioluminescence: Che mical Principles and Method s

ph oto pro tein has been developed for the biolum inescent proteins,

such as aequorin and obelin, that do not fit well with the definition of

luciferin or luciferase, as already mentioned. It is surprising that many

marine luminous organisms have been found to involve the identi

cal luciferin, coelenterazine. In addition, i t has been shown that the

reaction mechanisms of the luminescence reactions of firefly luciferin,

Cypridina

luciferin and coelenterazine all involve the same type of

interm edia tes, diox etan es, possessing a four-m em ber ring th at consists

of two carbon atoms and two oxygen atoms. Many luciferases have

been purified and characterized. Helped by the advances in genetic

technology, some luciferases and apophotoproteins have been cloned

and their three-dimensional structures have been determined.

Despite the rema rkable progress m ade , how ever, the t rend show n

in the table reveals a fact that cannot be interpreted favorably, at least

to this author. In the third quarter of the 20th century, the struc

tures of five different kinds of new luciferins have been determined,

whereas, in the last quarter, only three structures, of which two are

nearly identical, have been determined. None has been determined in

the last decade of the century and thereafter, thus clearly indicating

a declining trend, in contradiction to the steady advances in analyt

ical techniques. The greatest cause for the decline seems to be the

shift of research interest from chemistry and biochemistry into genetic

biotechnology in the past 20 years.

Chemical Study of Bioluminescence in the Future

Bioluminescence still has many mysteries, which may yield many

further insights into natu re an d science. In bioluminescence reac tions,

a luciferin generates the energy for light emission when oxidized.

For that rea son, luciferin is the mo st imp orta nt elem ent in biolum ines

cence; i t can be considered as the he art of the biolum inescence reaction .

Because of i ts importance, the author believes that the determination

and identification of the structure of luciferins should be considered

as one of the top targets in future research. The functional group of

a photoprotein corresponds to a luciferin in its function, thus it is as

important as a luciferin. Many luciferins and the functional groups

of photoproteins remain to be determined, and at least two of them


25/498

Introduction xxv

have been ready for structural work for many years: the luciferin of

the Bermuda fireworm Odontosyllis and the functional chrom op ho re

of the photoprotein pholasin from the clam Pholas. Each of these

two subjects has been briefly taken up from time to time at various

laboratories during the past 30 years , but no s tructural information

has been obtained for either of them.

Another important subject is related to the fashions of util izing

coelenterazine in bioluminescence reactions. Coelenterazine is widely

distributed in marine organisms and plays a central role in many

bioluminescent organisms. The compound is util ized at least in four

different fashions: (1) unmodified form, as luciferin in many organ

isms;

(2) disulfate form, as luciferin in the squid Watasenia; (3) per-

oxidized form, as the functional group of aequorin and obelin; and

(4) dehydro-form, for regenerating the squid photoprotein symplectin.

Many bioluminescent organisms contain substantial amounts of coe

lenterazine. However, some of them contain very weak luciferase

activities and their utilization of coelenterazine d o no t matc h with any

of the four cases given ab ov e. Th e exam ples of such organism s are: the

shr imp Sergestes, the squids Cbiroteuthis, an d the deep-sea fish Neo-

scopelus. In these orga nism s, coelenterazine m ight be util ized in o ther,

s t i l l unknown fashions. The s tudy of this l ine would be important to

fully understand the role of coelenterazine in bioluminescence.

In the luminescence systems that require a peroxide or an active

oxygen species in addition to molecular oxygen (the scaleworm, the

tube worm Chaetopterus, the clam Pholas, the squid Symplecto-

teuthis), theirin vitrolum inescence reactions reporte d are m uch slow er

and inefficient compared to their bright in vivo luminescence. The

true,

intrinsic activation factor in their in vivo luminescence should

be determined, and the detailed mechanisms of oxidation should be

elucidated.

Discovery of a new luciferin and a new mechanism will provide

us with enormous benefit , as i t was shown in the past. The work

may not be easy; however, the author believes that i t can be accom

plished when the researcher has a firm determination to complete

it . There is no established method or protocol for studying a new

type of lucifer in or photoprotein; thus, the method must be worked


26/498

xxvi Bioluminescence: Che mica l Principles and Me thods

out. Some suggestions on experimental procedures are included in the

Appendix.

The Contentsofthis Book

This book is devoted to the progress in the chemical under

standing of bioluminescence, particularly the mechanisms involved in

the light-emitting reactions. Though light emission from a luminous

organism may involve a series of biochemical reactions integrated in

a com plex m an ne r, the discussion in this boo k is focused on the light-

emitting reaction, i .e. the chemical reaction that results in the emis

sion of photons. The accessory reactions, such as the formation of

luciferin from preluciferin, the biosynthesis of luciferin, and the reac

tions involved in nervous stimulation, are not discussed unless they

are essential.

Th e me tho ds of the isolation an d purification of variou s luciferins,

luciferases an d ph oto pro tein s are described in detail as m uch as possi

ble because of their imp ortan ce. The re have been considerab le cha nges

in the methods, techniques and materials used during the 50-year span

covered by this bo ok . Ho w ev er, the unde rlying principles of the purifi

cation methods have not changed significantly; the old methods and

techniques are often very useful for the present research when the

principles involved are understood.

Th e future is an exten sion of the pas t. Th e au tho r believes th at th e

process of the prog ress m ad e in the pas t is as imp orta nt as the findings

and discoveries for the planning of future research. For this reason,

a substantial weight is placed on describing historical accounts. Such

inform ation w ou ld also help researchers to get some idea of the effort

that might be needed to isolate and identify a new luciferin.

Th e chapters in this boo k are arrange d roughly in the chron ologi

cal order of bioluminescence systems discovered, based on the date of

the major breakthrough made in each bioluminescence system, such

as the discovery of ATP in the firefly system (McElroy, 1947) and the

identification of fatty aldehyde as the luciferin in luminous bacteria

(Cormier and Strehler, 1953). This differs from Harvey's 1952 book,

which is arranged in the order of taxonomic classification.


27/498

Introduction xxvii

The description of luminous bacteria (Chapter 2) should be con

sidered minimal because the works related to this subject are too

enormous to be covered in this book; readers are referred to the

excellent review articles by Hastings and Nealson (1977); Ziegler and

Baldwin (1981);- Hastings et al. (1985); Hast ings (1986); Meighen

and Du nlap (1993); Tu an d Mag er (199 5); and Du nlap and Kita-

Tsukamoto (2001). Bioluminescence due to the presence of symbi

otic luminous bacteria is not covered in this book for the reason that

the chemistry involved is identical to that of luminous bacteria. The

reports of the data and information that are erroneous or irrelevant

to the objectives of this book are not cited, except when considered

essential, and reports of confirming nature are often omitted. For the

general topics of bioluminescenc e,Bioluminescence by Ha rvey (1952)

andBioluminescence in Action edited by H erring (19 78) are m ost use

ful even today, and are highly recommended. The references cited in

the text an d som e add itiona l relevant references are given in a lph abe t

ical order at the end of the book. For the convenience of researchers,

some basic data and information that might be useful are included in

the Appendix.


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29/498

C H A P T E R

T

THE FIREFLIES A N D LU MIN O U S INSECTS

Since ancient times, the light emitted by fireflies and glow-worms

has attracted the curiosity of people. Descriptions of the phenomena

are frequently found in old poems, songs and folklores of many coun

tries. Old scientific studies of these phenomena are also numerous,

particularly after the 17th century. However, the chemical study was

not begun until the early 20th century.

Although the class Insecta ( in the phylum Arthropoda) contains

bioluminescent organisms in four orders: Collembola, Hemiptera,

Coleoptera and Diptera, biochemical studies have been carried out

only w ith several types of organ isms of the last tw o ord ers, Co leopte ra

and Diptera. In these orders, the adults have two pairs of wings: in

Coleoptera, the front wings are modified as elytra (a heavy protec

tive cover); and in Diptera, the hind wings are reduced to knobs. The

order Coleoptera includes Lampyridae (fireflies), Phengodidae (rail

road worms), and Elateroidae (click beetles such asPyrophorus), and

all the lum ino us spec ies in this ord er utilize firefly luciferin in the ir ligh t

emission. The o rder Diptera co ntains the glow-w orms Arachnocampa

1


30/498


31/498

The F i re f l ies an d Lum inou s Insec ts 3

J 3

Fig. 1.1 T he firefly Luciola cruciata (male) draw n by Sakyo Kanda (1 87 4-1 93 9) , a

pioneer of the study of bioluminesccnce in Japan, showing his extraordinary artistic

talent (reproduced from Kanda, 1935).

1.1 Th e Fireflies

1.1.1 An Overv iew o fthe Firefly Luminescence React ion

The luciferin-luciferase reaction of fireflies was first demonstrated

by Harvey (1917), although the light observed was weak and short-

lasting. Thirty years after Harvey's discovery, McElroy (1947) made

a crucial breakthrough in the study of firefly bioluminescence. He

found that the light-emitting reaction requires ATP as a cofactor.

The addition of ATP to the mixtures of luciferin and luciferase


32/498


33/498

The F i re f l ies an d Lu min ous Insects 5

The following schemes represent the overall reaction of firefly bio-

luminescence (McElroy and DeLuca, 1978), where E is luciferase;

LH2 is D-luciferin; PP is py rop ho sp ha te; A M P is aden osine ph osp ha te;

LH2-AMP is D-luciferyl adenylate (an anhydride formed between the

carboxyl group of lucifer in and the phosphate group of AMP); and L

is oxyluciferin.

E + LH

2

+ ATP + Mg

2

+ E L H

2

- A M P + PP + M g

2 +

(1)

E L H

2

- A M P + 0

2

E L + C 0

2

+ A M P + Light (2)

In the first step , luciferin is co nv erted in to luciferyl ad en yla te by AT P in

the presence of M g

2 +

. In the secon d ste p, luciferyl ad en ylate is oxidized

by molecular oxygen resulting in the emission of yellow-green light,

of which the mechanism is discussed in Sections 1.1.6 and 1.1.7.Both

steps, (1) and (2), are catalyzed by luciferase. The reaction of the first

step is slower than that of the second step, thus the first step is the

rate-limiting step.

1.1.2 Firefly Luc iferin and O xyluciferin

Extraction and purification of luciferin. In the work of purifying

and crystallizing firefly luciferin (Bitler and McElroy, 1957), McElroy

used a unique method to gather the large quantity of fireflies needed

for their research. In the now legendary story, they advertised for the

purchase of fireflies at one cent per specimen. Children and youths in

the neighborhood responded enthusiast ical ly, col lect ing a huge num

ber of the bu gs for the m . In this w ay , they easily ob tain ed sufficient

number of the firefly Photinus pyralis for their research.

The live fireflies are dried over calcium chloride in a vacuum

desiccator, and then their lanterns are separated by hand. An acetone

powder prepared from the dried lanterns is extracted with boiling

w ater. The cooled aque ou s extract is extracted w ith ethyl acetate at p H

3.0, and the ethyl acetate layer is con cen trated un der redu ced pressure.

The concentrated luciferin is adsorbed on a column of Celite-Fuller 's

earth mixture. The column is washed with water-saturated ethyl

acetate, and eluted with alkal ine water at pH 8.0-8.5. The aqueous

eluate of luciferin is adjusted to p H 3.0 w ith H C1 an d luciferin is


34/498


35/498

The Fireflies an d Lum inous Insects 7

250 300 350

Wavelength (nm)

400

450

Fig. 1.3 A bs orp tio n spectra of firefly luciferin at p H 7.0 or below (solid line, A

m ax

32 7- 32 8 nm) and at pH higher than 9.0 (dashed l ine, A

m a x

3 81 -38 4 nm) . Reproduced

from McElroy and Seliger,

1 9 6 1 ,

with permission from the Joh ns Ho pk ins U niversity

Press.

of luciferin was accomplished by White

et al.

(1961; 1963); certain

details and the improvements of the synthetic method are discussed

by Bowie (1978) and Branchini (2000).

Oxyluciferin. Firefly oxyluciferin is an extremely unstable com

pound; i t has never been isolated in a completely pure form (White

and Roswell , 1991). A group in Nagoya synthesized the compound

and its properties were investigated (Suzuki

et al., 1969;

Suzuki and

G o to ,

19 71 ). Th e fluorescence of oxyluciferin in DM SO in vacuu m in

the presence of potassium -butoxide is yellow-green (A

max

55 7 nm ),

the same emission maximum as the chemiluminescence of luciferin in

DMSO in the presence of potassium -butoxide, suggesting that oxy

luciferin is the light emitter in the chemiluminescence of luciferin. In

the bioluminescence reaction, the absorption peak of synthetic oxylu

ciferin at pH 7 (382 nm; Suzuki

et al.,

1969) closely coincides with the

absorption peak of the luciferase-oxyluciferin complex in the spent

luminescence solution (Fig. 1.4; G ates and D eLu ca, 1 97 5), suggesting

that oxyluciferin is the light-emitter as in the case of chemilumines

cence. However, the fluorescence emission maximum of the spent


36/498

8 B i o l u m i n e s c e n c e : C h e m i c a l P r in c ip l es a n d M e t h o d s

0.5 -r

0.4 -

(D

O 0.3 -

C D

o


37/498


38/498


39/498

The F i re f l ies an d Lu m ino us Insec ts 11

1.1.4 Assayso fLuc iferase Ac tivity, ATP and Luc iferin

Various assay mixtures of different compositions have been used

to measure the activity of luciferase and the amount of ATP (Leach,

198 1). A typical m ixtu re for luciferase assay contains 1 0 -2 5 m M

Tris-HC l or glycylglycine buffer, p H 7. 5- 7. 8, 5 m M M g C l

2

, 1-5 mM

ATP,

and 0 .1 m M luciferin. Because luciferase at very low c once n

tra t ions is rapidly inactivated, 0 .5-1 m M EDTA and 0 . 1 % BSA are

included in some formulae to prevent the inactivation. Usually ATP

is injected into the rest of the mixture to start luminescence, pro

ducing a sharp flash of light that diminishes rapidly (DeLuca and

McElroy, 1978). The peak of the f lash occurs about 0.3-0.5 second

after the injection of ATP (Fig. 1.6), and the light intensity of the peak

is pr op ort ion al t o the am ou nt of luciferase in a wide rang e of luciferase

concentration. If the measurement of flash height is difficult to carry

out for some reasons (such as a slow response of recorder), the light

intensity at 5 or 10 secon ds after the AT P injection is m easu red instead

of the flash height. Although the measured light intensity in this case

10.0

T

9.0 -

8.0

7.0 -

6.0 -

tfi

J j

4

' -

c

g 3.0 -


40/498

12 Bioluminescence: Chem ical Principles and Methods

is lower than the intensity of a flash, it is still proportional to the

amount of luciferase as long as the same method and the same con

ditions are used. Luciferin and ATP can be assayed with appropriate

modifications of the method.

1.1.5

Genera l

Characteristics

o fthe Bioluminescenceo fF ireflies

The color of the luminescence of common fireflies varies slightly

depending on the species, with their in vivo emission peaks in a range

from 55 2 nm to 5 82 nm (Seliger and M cElroy , 19 64 ). Th e color of

thein vitro luminescence using purified luciferin and Pho tinus pyralis

luciferase (plus ATP and Mg

2 +

) under neutral or slightly alkaline

conditions is yellow-green (A

max

56 0 nm ; Fig. 1.7), w ith a qu an tum

yield of 0.88 0.25 (Seliger and McElroy, 1959; 1960); in spite of the

large error range, the quantum yield is clearly greater than those of

Cypridina luciferin and coelenterazine (both about 0.3). The quantum

yield and the color of luminescence are affected by the pH of the reac

tion medium (Fig. 1.8). Under acidic conditions, red luminescence

( max 6 15 nm ) w ith a decrea sed light intensity is em itted. A simi

lar red shift of luminescence is also observed by raising the reaction

120 j

100 -

% 8 0 -

c

03

1 6 0 -

>

20 -

I i

500 550 600 650 700

Wavelength (nm)

Fig. 1.7 Spectral chan ge of the

in vitro

firefly bioluminescence by pH, with

Photinus pyralis luciferase in glycylglycine buffer. The normally yellow-green lumi

nescence (A.

max

560 nm) is changed into red (A

m ax

615 nm) in acidic med ium, accom

panied by a reduction in the quantum yield. From McElroy and Seliger, 1961, with

permission from Elsevier.


41/498


42/498


> >

%

8 0 -

c

CD

L

g

n

R

e

a

v

e

O

O

0 -

/ " \

/ \

/ \

10 20 30

Temperature (

c

C)

40

Fig. 1.10

Effect of tem pe ratu re on the activity of luciferase. Fro m M cElroy a nd

Seliger, 1 9 6 1 , with pe rmission from Elsevier.

(Fig. 1.9; Green and McElroy, 1956). The optimum temperature for

luminescence is 23-25C (Fig. 1.10; McElroy and Strehler, 1949;

Green and McElroy, 1956). In the presence of a low concentration

of ATP, high concentrations of M g

2 +

are inhibitory , while in the pres

ence of a low concentration of Mg

+

, high concentrations of ATP are

inhibitory (Fig. 1.11; Green and McElroy, 1956).

>

to

0

2 0

o -

/ /

/ /

j /

/

/

l

l

l

J

'

-

--*'''"""

^ 1 .6 m M A T P

/

/

~ ~ .

0.16 mM ATP

i 1 1 1 1 1 1 1 1 1 1 1 1

Concentrat ion of Mg

2+

(mM)

Fig. 1.11

Effect of M g

2 +

concentration on the activity of luciferase in the presence

of 0 . 16 m M and 1.6m M ATP. From Green and McElroy, 1956 , wi th permission

from Elsevier.


43/498

The Fireflies and Lum inous Insects 15

The sharp flash in the firefly bioluminescence reaction (Fig. 1.6) is

due to the formation of a strongly inhibitory byproduct in the reac

tion. The inhibitor formed is dehydroluciferyl adenylate, having the

structure shown below at left. In the presence of coenzyme A (CoA),

however, this inhibitory adenylate is converted into dehydroluciferyl-

CoA, a compound only weakly inhibitory to luminescence. Thus, an

addition of CoA in the reaction medium results in a long-lasting, high

level of luminescence (Airth et al., 1958; McElroy and Seliger, 1966;

Ford et al, 199 5; Fontes et al., 1997, 1998) .

^ \ ^-N tvL /C O-A MP ^ \ ^ - N | \ L / X O - C o A

f T w T

+COA^> fXy^X

+

^

1.1.6 Mechanisms

o f

the Firefly Bioluminescence

Seliger and McElroy (1962) discovered that the esters of firefly

luciferin emit chemiluminescence. They reported that luciferyl adeny

late (Rhodes and McElroy, 1958) emitted a red light (A.

max

625.5 nm)

in dimethyl sulfoxide upon the addition of a base. The emission spec

trum was dependent upon pH, producing a yellow-green l ight in the

presence of a large excess of base. The observation of yellow-green

light was also reported later by other authors (White et al., 1969,

1971;

however, see Section

1.1.7).

The pr odu ct of the luminescent oxi

da tio n of luciferin is oxyluciferin (the stru ctu re sh ow n in Fig.

1.12),

an

extremely unstable compound. Hopkins et al. (1967) found that 5,5-

dimethyloxylucferin, an oxyluciferin analogue having no H atoms at

position 5, shows a red fluorescence in the presence of a base, coincid

ing with the red chemiluminescence spectrum of luciferyl adenylate.

Co nside ring these findings, the biolum inescence reac tion of firefly w as

postulated as show n inFig. 1.12 (Hopkinset al., 1 9 6 7 ; M c C a p ra etal.,

1968;

Whi te et al., 1969 , 1971 , 1975; Sh imomura et al., 1977; Koo

etal., 1978).

In the postulated bioluminescence mechanism, firefly luciferin is

adeny lated in the presence of luciferase, ATP and M g

2 +

. Luciferyl

ade ny late in the active site of luciferase is quickly oxy gen ated at its ter

tiary carbon (position 4), forming a hydroperoxide intermediate (A).


44/498

16 Bioluminescence: Che mical Principles and Methods

COOH

4

ATP

a

Adenylate

N

D-Luciferin

AMP

j Xf

Oxyluciferin C1

(Red light)

OR

O

Oxyluciferin C2

(Yello-green light)

CO-AMP

+ PP

O O H

- A M P

Fig. 1.12 M echa nism of the biolum inescence reaction of firefly luciferin catalyzed by

firefly luciferase. Luciferin is probably in the dianion form when bound to luciferase.

Luciferase-bound luciferin is converted into an adenylate in the presence of ATP and

M g

2 +

, splitting off pyrophosphate (PP). The adenylate is oxygenated in the presence

of oxygen (air) forming a peroxid e intermediate A, which forms a dioxetan one inter

mediate B by spli t t ing off AMP. The decomposition of intermediate B produces the

excited state of oxyluciferin m on oa nio n (C I) or dian ion (C2). W hen the energy levels

of the excited states fall to the gro un d s tates, CI and C2 emit red light (A.

max

615 nm)

and yellow-green light

(X

max

560 nm), respectively.

The hydroperoxide forms a very unstable 4-membered dioxetanone

ring (B), split t ing off AMP. The dioxetanone decomposes by a con

certed cleavage, yielding the keto-form oxyluciferin (CI) and CO2,

accompanied by emission of l ight. I t is possible that the decomposi

tion of dioxetanone results in light emission by the chemically initi

ated electron-exchange luminescence (CIEEL) mechanism (McCapra,

1977; K oo

et ai,

197 8). The formation of the dioxetan one interme

diate was confirmed by

l s

O-labeling experiments , by showing that

one of the O atoms of the product CO2 was derived from molecular

oxygen (Shimomura

et ai,

1 97 7; also see Section 1.1.8).


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18 Bioluminescence: Chem ical Principles and Method s

M c C a p r a

et al.

(1994) and McCapra (1997) suggested that the

color variation could be caused by the conformational difference of

the oxyluciferin molecule, when the plane of thiazolinone is rotated

at various angles against the plane of benzothiazole on the axis of the

2-2 '

bond; the red light would be emitted at 90 angle, reflecting its

minimum structural energy.

However, Branchini et al. (2002) reported a surprising discovery

that the adenylate of D-5,5-dimethylluciferin emits light in two differ

ent colors in the bioluminescence reaction catalyzed by two different

luciferases, one from Photinus pyralis and the other from a green-

emitting click beetle Pyrophorus plagiophthalamus. In the presence

of Mg

2 +

and at pH 8.6, a yellow-green light (A

max

5 60 nm) was pro

duced with P. pyralis luciferase and a red light (A

max

624 nm) was

emitted with P. plagiophthalamus luciferase. In bo th cases, the reac

tion pro du ct was 5,5-dimethyloxyluciferin (shown below) tha t has no

H ato m on its C5 ; it ca nn ot take the tautom eric enolized form , such as

in C2, C3 or C4, that had been proposed to be the emitter of yellow-

green light.

s s -^v"

C H 3

C H

3

This finding by Branchini

et al.

(2002) clearly indicates that 5,5-

dim ethyloxyluc iferin is able to emit the tw o different c olo rs. This con

clusion, however, does not rule out the involvement of the enolized

oxyluciferin in the bioluminescence reaction of firefly.

Orlova

et al.

(2003) theoretically studied the mechanism of the

firefly biolum inescen ce reac tion on the basis of the hybri d density func

tional theory. According to their conclusion, changes in the color of

light emission by rotating the two rings on the 2-2' axis is unlikely,

wh ereas the partic ipatio n of the enol-forms of oxyluciferin in biolumi

nescence is plausible but not essential to explain the multicolor emis

sion. They predicted that the color of the bioluminescence depends

on the pola rization of the oxyluciferin m olecule (at its O H an d O

termini) in the microenvironment of the luciferase active site; the

XX


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smaller the H - O po larizatio n, the greater the blue shift of the ab sorp

tion (and ex citation). By this m ech anism , the rang e of colors o bserved

in the bioluminescence could be obtaine d w ith various forms of oxylu

ciferin. Th e mo st likely light emitter is keto-s-tran s m on oa nio n, b ut the

enol-s- trans m on oa nion and keto-s-cis m on oan ion structures may also

be involved. Their conclusion is in agreement with that of Branchini

et al. (2002), in that the involvement of keto-enol tautomerism is not

essential to explain the two different colors.

According to Branchini et al. (2004), luciferase modulates the

emission color by control l ing the resonance-based charge dereal iza

tion of the anionic keto-form of oxyluciferin in the excited state. They

proposed the structure C5 as the yellow-green light emitter, and the

structure C6 as the red light emitter.

C5 C6

(yellow-green) (red)

It should be pointed out that the structure C5 (yellow-green emit

ter) is identical to the structure CI that was previously assigned to the

red light emitter.

1.1.8

ANote on the D ioxetanone Pathw ay and the

X8

0-incorporat ion Experiment

In the luminescence reaction of firefly luciferin (Fig. 1.12), one

oxygen a tom of the pro du ct CO 2 is derived from the molecular oxygen

while the other originates from the carboxyl group of luciferin. In

the chemiluminescence reaction of an analogue of firefly luciferin in

DMSO in the presence of a base, the analysis of the product CO2 has

supported the dioxetanone pathway (White et al., 1975).

Contrary to the dioxetanone pathway, DeLuca and Dempsey

(1970) proposed a mechanism of the bioluminescence reaction

that involves a multiple linear bond cleavage of luciferin peroxide


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However, the linear bond cleavage hypothesis of the firefly bio-

luminescence was made invalid in 1977. It was clearly shown by

Shimomura et al. (1977) that one O atom of the CO2 produced is

derived from molecular oxygen, not from the solvent water, using the

same

lg

O-labeling technique as used by DeLuca and Dempsey. The

result was verified by Wannlund et al. (1978). Thus it was confirmed

that the firefly bioluminescence reaction involves the dioxetanone

pathway. Incidentally, there is currently no known bioluminescence

system that involves a splitting of CO2 by the linear bond cleavage

mechanism.

It seems important to identify the factors that have led DeLuca,

Dem psey, and others into a misjudgment. The following explan ation

is included here for future experimentalists (see also Section C6 in

Appendix).

W he n gaseous CO2 is equilibrated w ith aque ous buffer solution in

a closed vessel, a large portion of the CO2 is dissolved in the aqueous

phase, mostly in the form of bicarbonate, maintaining the equilibrium

of the following three phases:

C 0

2

+ H

2

0 < H2CO3 * H C O J + H+

T h u s , the O atom of CO2 is exchangeable with the O atom of H2O.

When the luminescence reaction is carried out in a H2

1 8

0 medium

under an atmosphere of

16

Oi, the C

16

C>2 formed by the dioxetanone

mechanism is spontaneously converted into C

1 6

0

1 8

0 . If the rea ction

is carried out in a H

2

1 6

0 medium under an atmosphere of

18

C>2,

the C

1 6

0

1 8

0 formed is spon taneou sly converted into C

16

C>2. Thus,

the result of

l s

O-incorporation experiment can be obscurred by the

exchange of O atom between CO2 and H2O. In addition to this

exchange, the presence of contaminating CO2 can also obscure the

result. The occurrence of CO2 is ubiquitous and clean air normally

contains appro xim ately 0 .0 3 % (v/v) of C O2. In our experimen ts, care

fully prepared fresh buffer solutions contained 0.02-0.03 umol/ml of

CO2 plus HCO^ even after vacuum degassing, and the amount was

much greater when luciferase had been included (Shimomura et al.,

1977) . Thus, the CO2 produced from a small amount of luciferin (for

example, 0.033 umol in 3.5 ml: DeLuca and Dempsey, 1970) will be


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organs located at the posterior extremity. They are found most often

on the roofs of caves. The glow-worms usually stay on the horizontal

network of mucous tubes suspended from rocks, and they hang down

long sticky thre ad s of fishing line s from the tub es to catc h small

insects. The spectacular view of glow-worms at the Waitomo Cave in

New Zealand at tracts hundreds of touris ts everyday.

Earlier studies indicated that the bioluminescence emission max

imum of the New Zealand glow-worm

A. luminosa

is 48 7-4 88 nm ,

and that the bioluminescence reaction probably requires ATP as a

cofactor (Shimomura et al., 1966), similar to the firefly luminescence

reaction. According to Lee (1976), the luminescence emission spec

trum of the Austral ian glow-worm A. richardsae (A

max

4 88 nm) was

n o t significantly influenced by p H in a w ide ran ge , i.e. 5 .9 -8 .5 , and fire

fly luciferin does not cross-react with the spent luminescence mixture,

indicating differences from the firefly luminescence system. However,

the luminescence was quenched by ED TA , but not by EG TA th at does

not chelate Mg

2 +

, suggesting that Mg

2 +

is probably required for the

luminescence reaction, like the firefly luminescence.

The luciferin-luciferase reaction of Aracbnocampa w as first

demonstrated by Wood (1993), by mixing a cold-water extract and a

cooled hot-water extract . The cold-water extract was prepared with

27 m M Tricine, pH 7.4, containing 7 m M MgSC>4, 0.2 m M ED TA ,

1 0 %

glycerol and 1% Tri ton X-100, and incubated with 1m M ATP

on ice for 18 hr. The ho t-w ater e xtrac t was prep ared by heating the

cold w ater e xtract before the a ddition of AT P at 98C for 5 m in. The

luminescence reaction was performed in the presence of

1

m M ATP .

Extraction and purification of luciferin and luciferase (Viviani

etal.

2002a)

T o isolate luciferin, the lantern s of the Au stralian

A. flava

were homogenized in hot 0.1 M citrate buffer, pH 5, and the mixture

w as heated to 95C for 5 m in. The mix ture was acidified to pH 2. 5- 3. 0

w ith HC 1, and luciferin w as extracted w ith ethyl acetate. U po n thin-

layer chromatography (ethanol-ethyl acetate-water, 5:3:2 or 3:5:2),

the active fraction of luciferin was fluorescent in purple (emission

A-max 4 15 nm w he n exc ited at 2 90 nm ). To isolate the luciferase, the

cold-water extract prepared according to Wood

(1993;

see above)

was chromatographed on a column of Sephacryl S-300. On the same


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The Fireflies and Luminous Insects 29

Th e hot-wa ter extract was prep ared by heat ing the cold-water e xtract

at 95C for 5 -1 0 min in the presence of lO m M D TT unde r argon

gas. Th e luminescence react ion w as performed in 0.1 M Tris-HC l,

pH 8.0. The react ion was s trongly s t imulated by DTT and ascorbic

acid, but not by ATP, indicating that theOrfelia lumine scence sys

tem is different from the luminescence systems of the fireflies and

Arachnocampa th at require AT P for light em ission. After the lum ines

cence of a cold-w ater extrac t in the p H 8.0 buffer co ntainin g D T T h ad

decayed to ab ou t 10 % of the peak intensity, an add ition of a hot-w ater

extract caused an immediate increase in light emission (Fig.

1.17),

sug

gesting that the decay of luminescence is caused by the depletion of

luciferin. The molecular mass of the luciferase was estimated at about

140 kDa by gel fi l tration. The luminescence of O. fultoni is the blue st

of all luminous insects (A.

m ax

460nm; Fig.

1.16).


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Luminous Bacteria 33

(reviews: Hast ings and Nealson, 1977; Ziegler and Baldwin, 1981;

Hastings et al, 19 85 ; Baldwin and Ziegler , 199 2; M eighen and

D un lap , 199 3; Tu an d Ma ger, 1 995 ). The publicat ions relating to

bacterial bioluminescence reaction are so numerous that i t is not pos

sible to go into detail in this book. Only a brief outline is given in the

following sections.

2.2 B acterial Lu ciferase

Cultivation of luminous bacteria. Nealson (1978) lists various

culture media to culture lumin ous bacte ria. Th ree exam ples from othe r

sources are show n in Tab le

2 .1 .

I t is im po rtant to include 30 0 -5 0 0 m M

NaCl as a basic ingredient. For the growth of bacteria, l iquid media

must be adequately aerated by shaking or bubbling. Solid media con

taining agar are made in Petri dishes.

Tab le 2.1 Exam ples of Culture M edia for Grow ing Lum inous Bacteria

Seawater

Complete

NaCl

Complete

3

Solid

Medium

b

Dist. water

Sea water

NaCl

N a

2

H P 0

4

- 7 H

2

0

K H

2

P 0

4

( N H

4

)

2

H P 0

4

MgSC-4

C a S 0

4

Glycerol

Tryptone

Yeast extract

Agar

Bacto-nutrient agar (Difco)

Temperature

200 m l

800 ml

2 ml

5 g

3 g

(10 -20 g)

30-34C for

B.

harveyi

1,000 ml

3 0 g

7 g

l g

0.5 g

0.1 g

2 ml

H

3 g

(10 -20 g)

26C for

P.

fischeri

1 8 C fo r

P. phosphoreum

1,000 ml

3 0 g

5 g

10 ml

30

g

25C

a

From Has t ings et al., 1978 ; add 10 -20 g agar to make sol id m edium.

b

From Shimomura et al, 1974a.


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3 6 B i o l u m i n e s c e n c e : C h e m i c a l P r in c ip l es a n d M e t h o d s

Tab le 2.2 Am ounts of Saturated Fatty Aldehyde Extracted from 4 0 ;

of Luminous Bacteria (Shimomura

et al.,

1974a)

Luminescence intensity

of bacterial suspension,

1 mg/ml, a t 20C

(quanta/s per ml)

Total amount of fatty

aldehydes (nmol)

Fatty aldehyde (nmol) of

10-carbon

11-carbon

12-carbon

13-carbon

14-carbon

15-carbon

16-carbon

17-carbon

18-carbon

P. phospboreum

6 x 10

1 2

600

< 1

< 1

30 (5%)

6 (1%)

3 80

(63%)

6 (1%)

180

(30%)

< 1

< 1

A. fischeri

1.1 x 10

1 2

90

< 1

< 1

32 (36%)

2 (2%)

29 (32%)

6 (7%)

18 (20%)

2 (2%)

< 1

A. fischeri

Aldehydeless

Dark Mutant

1 x 10

8

7

< 1

< 1

1.5 (22%)

< 1

0.9 (13%)

0.5 (7%)

3.8 (54%)

0.14 (2%)

0 . 0 7 ( 1 % )

P. phospboreum.

In the case of

A. fischeri,

the total amount of alde

hydes was only 15% of that from

P. phosphoreum,

wh ich consisted

of dodecanal (36 % ), te tradecanal (32% ) and hexadecanal (2 0% ). The

contents of aldehydes having the carbon atoms of 10, 11, 13, 15, 17

and 18 were negligibly small in both bacterial species.

According to the data in Table 2.2, 1 g of P.

phosphoreum

cells

continuously emit 6 x 10

1 5

ph oton s/s at a cell co nce ntration of1g/liter

at 20 C, w herea s the am ou nt of total aldehydes o btaine d from the

same cells ( lg ) is 15 nm ol. This am ou nt of aldehydes is capab le of

emitting a total light of 1.44 x 10

1 5

photons , assuming the quan

tum yield of the aldehydes (see Section 2.6) to be 0.16. Considering

that the light emission from luminous bacteria is continuous and in

a steady-state, and that the overall yield of the aldehydes is probably

5 0 - 7 0 %

, these figures indicate that bacterial cells contain an amount

of aldehydes that can sustain the luminescence for only about

0.3-0.5 s, suggesting that the long-chain aldehydes are continuously


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38 Bioluminesce nce: Che mica l Principles and Me thods

+ H

2

0 + Light

u

Fig. 2.1 M echan ism of the bacterial bioluminescence reaction. The molecule of

FM N H 2 is dep roton ated at N l wh en bo und to a luciferase m olecule, which is then

readily peroxidized at C4a to form Intermediate A. Intermediate A reacts with a fatty

aldehyde (such as dodecanal and tetradecanal) to form Intermediate B. Intermedi

ate B decomposes and yields the excited state of 4a-hydroxyflavin (Intermediate C)

and a fatty acid. Light (A

m ax

4 90 nm ) is em itted wh en th e excited state of C falls

to the ground state. The ground state C decomposes into FMN plus H2O. All the

intermediates (A, B, and C) are luciferase-bound forms. The FMN formed can be

reduced to FMNH2 in the presence of FMN reductase and NADH.

The deprotonated flavin in the complex is readily attacked by molec

ular oxygen at C4a, giving 4a-hydroperoxide of the flavin-luciferase

complex (intermediate A). This complex is an unusually stable inter

m ed iate, with a l ifetime of tens of seconds at 20 C and ho urs a t sub

zero temperatures, allowing its isolation and characterization (Hast

ingset al., 19 73; Tu, 197 9; Balny and H ast ings , 19 75; Vervoor t et al.,

1986;

K urfuerst et al., 1987; Leeet al., 1988) .

In the presence of a long-chain fatty aldehyde, intermediate A

(Fig. 2.1) is conv erted in to interm ediate B tha t con tains a p ero x-

yhemiacetal of flavin (Macheroux et al., 1993) . The apparent K

m

value of the intermediate B produ ced with an aldehyde of 10 -1 3

carbon atoms is approximately 200 uM whe n P. pbospboreum

luciferase is used (Watanabe and Nakamura, 1972), and 1-10 uM

when P. fischeri luciferase is used (Spudich and Hastings, 1963;

Hast ings and Nealson, 1977). The decomposit ion of intermediate B,


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Luminous Bacteria 39

through several steps, yields the excited state of 4a-hydroxyflavin-

luciferase complex (intermediate C) and a fatty acid. The mechanism

of the formation of the excited state possibly involves the chemically

initiated electron exchange luminescence (CIEEL) mechanism (Schus

ter, 1979). The light emitter is considered to be luciferase-bound 4a-

hydroxyflavin (intermediate C) (Ghisla et al., 1977; Kurfurst et al.,

1987;

Leiet al., 2004) .

2.5 Assay

of

Lu ciferase A ctivity (Has tingset a l ., 1978;

Baldwin

e t

a/. , 1986)

The activity of bacterial luciferase is measured by recording the

light intensity when 1ml of FM N H 2 solution is rapidly injected into

1 ml of 50 m M ph osp hate buffer , p H 7.0, containing 0 .1 -0 .2 % BSA,

0.1 m M long-chain aldehyde (decanal , dodec anal or tetradecanal) and

a luciferase sample, at 20-25C (Fig. 2.2). The peak light inten

sity is proportional to the amount of luciferase at a wide range of

enzyme concentrat ions. FMNH2 is extremely unstable under aerobic

Time (sec)

Fig. 2.2 Assay of luciferase by the injection of F M N H 2. The assay wa s initiated by

the injection of 1 ml of 50 |JLM FMNH2 solut ion in to 1ml of air equilibrated buffer,

pH 7.0, containing 0.1% BSA, luciferase, and 20 |JL1of 0. 0 1 % sonicated suspension

of dodecanal. From Baldwin et al., 1986, with permission from Elsevier.


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Lum inous Ba cteria 45

Petushkovet al, 19 95) . Both types of pro teins have m olecular w eights

of about 20,000 (Small et al., 1980). When a lumazine protein is

included in the bacterial in vitro bioluminescence reaction, the nor

m al 490 nm luminescence max im um is shifted to 47 6 nm , ac com pa

nied by a significant increase in the light output (Gast and Lee, 1978;

O'Kane and Lee, 1986). On the other hand, the blue-green biolumi

nescence emitted with the purified luciferase from P. fischeri strain

Y-l (A.

max

48 4 nm ) is shifted to yellow (A.

max

534 nm) on addition of a

YFP obtained from the same strain (Daubner etal., 1987; M acheroux

et al., 1987). These spectral shifts of light emission clearly show the

occurrence of energy transfer.

C H

2

O H C H

z

O - R

( CH O H )

3

( CH O H )

3

C H

2

C H

2

C H

3 ^ / N N O

C I V ^ NT

F M N R = P O ( O H )

2

Riboflavin R = H

The absorption maxima of the recombinant LumP-type proteins

from P. leiognathi that contain the prosthetic groups of lumazine,

F M N , an d riboflavin are at 4 20 nm , 458 nm and 46 3 nm , respec

tively. In these highly fluorescent proteins, the prosthetic groups

are t ightly bound to the apoprotein; their dissociation constants

are:

0 .26 nM with lumazine; 30 nM with F M N ; and 0 .53 n M with

riboflavin (Petushkov et al., 1995). In the case of YFP, the dissocia

tion constant of the FMN-protein binding is reported to be 0.4 nM

(Visser et al., 1997). When these fluorescent proteins are bound with

the luciferase co m plex con taining the excited state of 4a-hydroxy flavin

(intermediate C; emission m axim um 49 0 nm ), a resonance energy

transfer takes place from the flavin to the fluorescent protein, result

ing in the appearance of new luminescence emission peaks, at about


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475 nm in the presence of LumP containing lumazineis and 540 nm in

the presence of LumPs containing FMN or riboflavin.

Th e spe ctral shift in the latter ca se, from 4 90 nm to 5 40 nm , is

readily understandable because of a good overlap between the emis

sion spectrum of the intermediate C and the absorption spectrum of

LumP-flavin (see Fig. 2.4). However, the former case of energy trans

fer, from 49 0 nm to 4 75 nm (Lee, 19 93 ), has a n a pp are nt difficulty

due to the energetics, because such a change requires an increase in

the energy level. According to Tu and Mager (1995), however, there

is a significant spectral overlap between the emission spectrum of C

and the absorption spectrum of LumP for an occurrence of the res

onance energy tansfer. The same authors also noted the possibili ty

of an energy transfer from an intermediate luciferase complex, which

precedes C and has an energy level higher than C, to the fluorescent

protein.

120

100 -

4


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T h e O s t r a c o d Cypridina (Va rgu lo ) an d o t he r Lum i nous Crus t ac eans 51

3.1.2 Cypridinahilgendorfii Miiller

This species is abundant in shal low waters around Japan and

Southe ast Asia. Th e org anism is egg-shaped an d abo ut 2 mm long

(Fig. 3.1.1). An individual animal contains roughly 1pg each of

luciferin and luciferase. C. hilgendorfii lives in sandy bottom close

to shore and comes out at night swimming around to feed on what

ever is available. This ostracod is a voracious scavenger. If a large

fish head tied to a string is thrown into the bottom of shallow water,

the ostracods would nibble at the bait . After about an hour, when

the bait is slowly pulled up to the surface, the tiny ostracods can be

washed off from the bait. If the bait is left at the bottom too long,

several hours or overnight, the ostracods would consume all the meat

and other edible parts, leaving only the bone and hard skin. Cyprid

ina has a large luminous gland consisting of gland cells of two kinds,

one secreting luciferin and the other secreting luciferase. The animal

squirts the luciferin and luciferase into the seawater in response to

a predator or stimulation. Mixing of these two substances will give

rise to a bright blue cloud of luminescence in the seawater, while the

Fig. 3.1.1 Th e ostra cod Cypridina hilgendorfii (photo by Dr. Tos hio Go to) .


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The Ostracod Cypridina (Vargula)and other Luminous Crustaceans 53

the southern Pacific. One of the intended uses was to mark the backs

of soldiers at night with the glowing substance, allowing soldiers to

identify and follow one another silently through the dark jungles. It

ap pe ars, how ever, tha t non e of the material wa s actually util ized in the

War, since some of the material was sunk with the transport ships by

US subm arines a nd the rest becam e quickly useless in the high hum id

ity of the tropical climate, probably due to poor desiccation. Harvey

died in 1 95 9, leaving a large am ou nt of dried Cypridina at his labora

tory in Princeton . Some bottles of theCypridina we re transferred to my

han ds throu gh D r. Aurin C hase wh en I w as in Princeton. The m aterial

is sti l l at my laboratory and shows a good luminescence when moist

ened with wate r even after m ore tha n half a century of storage at r oo m

temperature .

Cultivation. A num be r of

Documents

Bioluminescence Chemical Principles